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BCL2+IL6+ plasma cell tumors 1 Supplemental Methods 2 3 Mice 4 All studies were performed on the BALB/c (C) background and approved under IACUC Protocol 5 0006A56361. BCL2+IL6+ congenics harbor the EμSV-Bcl-2-22 1 and H2-Ld-IL6 2 transgenes. CD45.1+ 6 congenics carry the SJL-derived allotype of CD45, CD45.1, instead of the C allotype, CD45.2. 7 8 Adoptive B-cell transfer 9 Splenic B cells were obtained from 5-8 weeks old BCL2+IL6+ mice using the MACS B220 Mouse B-cell 10 kit (Miltenyi, Cambridge, MA). Two days later, 2 million of these cells were transferred to normal C host 11 mice, using retroocular intravenous (IV) injection. Prior to the adoptive transfer, B cells were 12 maintained in vitro in RPMI1640-based cell culture medium supplemented with 10% inactivated fetal 13 calf serum, 1% penicillin/ streptomycin, 200 M L-glutamine and 50 M 2-mercaptoethanol. B cells 14 were stimulated in culture (37 °C; 5% CO2) with 10 g/ml LPS and 12.5 ng/ml mIL-4. Two hours prior 15 to cell transfer, 2-4 months old hosts were conditioned with either 450 cGy whole-body irradiation 16 (sublethal dose not requiring HSC rescue) or 1,100 cGy whole-body irradiation (lethal dose requiring 17 HSC rescue). The latter was accomplished by transferring 3 x 106 normal bone marrow cells (obtained 18 from inbred C mice) together with the BCL2+IL6+ B cells. 19 20 DNA constructs and viral stock 21 All PCR products were cloned into pCRII-TOPO (Invitrogen) and confirmed by DNA sequencing. To 22 generate a retrovirus that co-expressed tGFP (turbo green fluorescence protein) and firefly luciferase 23 (fLuc), we cloned the respective cDNA genes into MSCV (murine stem cell virus). Using an intervening 24 picornoviral 2A segment to accommodate both genes, we thus produced MSCV-tGFP-2A-fLUC. All 25 DNA preparations were purified using Zymo Research DNA plasmid purification kits (Irvine, CA, USA). 26 Sequences and maps are available upon request. Preparation of the retrovirus was as follows: 293T 27 cells (Clontech Laboratories, Mountain View, CA, USA) were co-transfected with 15 μg retroviral vector 28 DNA and 15 μg Psi2 (2) packaging vector DNA, using the CaCl2 method. Medium was changed 4-6 29 hrs post transfection. Supernatant was harvested 48 hrs after transfection. Supernatant was filtered 30 using PVDF with a pore size of 0.45 μm. Virus was freshly prepared for each use. 31 32 Viral gene transduction 33 For retroviral reporter gene transduction, a spinfection protocol was used. Briefly, B cells were plated in 34 6-well cell culture dishes (3.5 × 106 cells per well) that contained one-third viral supernatant and two- Tompkins et al. – Supplemental Information 1 BCL2+IL6+ plasma cell tumors 35 thirds cell culture medium. The latter was supplemented with 7.5 g/ml polybrene. Plates were then 36 subjected to centrifugation (500 x g, 2 hrs, 30 C) followed by transfer of plates to ambient temperature 37 (30 min). Cells (still in viral supernatant) were then pelleted (300 x g, 10 min), re-suspended at 1 x 106 38 cells/ml in cell culture medium, and incubated overnight (37 °C; 5% CO2). The next morning, cells were 39 harvested and transferred to host mice as described above. Transduction efficiency, based on flow 40 cytometric evaluation of fluorescent reporter protein expression, was ≥90%. 41 42 Diagnosis and histopathology of plasma cell neoplasms (PCNs) 43 Incipient tumors were detected by monitoring mice for health status parameters, including occurrence 44 of hind limb weakness or paralysis. The diagnosis of PCN was established at necropsies of tumor- 45 bearing mice and confirmed histologically using criteria described in the Bethesda classification of 46 mouse hematopoietic tumors 3. Four- icrometer sections of paraffin-embedded tissues were stained 47 with hematoxylin and eosin (H&E) and evaluated by a board-certified hematopathologist (Carol 48 Holman). For detection and enumeration of osteoclasts in situ, the activity of tartrate-resistant acid 49 phosphatase (TRAP) was used as biomarker. Briefly, bone sections were fixed with formaldehyde 50 followed by decalcification in 10% EDTA for 6 days at 4 °C. The decalcified bones were rinsed with 51 PBS three times (3 x 5 min), postfixed in cold 70% ethanol, and embedded in paraffin. Tissue sections 52 were deparaffinized and stained for TRAP activity, using an acid phosphatase leukocyte kit from Sigma 53 according to the manufacture’s suggestions. Specimens were counter-stained with eosin and mounted 54 for imaging and histomorphometry. 55 56 Flow-cytometric analysis of PCNs 57 Antibodies to B220-PE-Cy7 (6B2), CD45.1-FITC (A20) and CD45.2-PE (104) were purchased from 58 eBioscience (San Diego, CA). Antibody to CD138-APC (281-2) was purchased from BD Biosciences 59 (San Jose, CA). To obtain single cell suspensions of lymphocytes, lymphoid tissues were harvested 60 and minced between frosted glass slides. ACK lysis (Lonza, Radnor, PA) was used to remove red 61 blood cells. For flow analysis, one million cells were washed and re-suspended in staining buffer that 62 consisted of balanced salt solution, 5% bovine calf serum and 0.1% sodium azide. Non-specific 63 binding of antibody was blocked using 10 μl rat serum (Jackson Immunoresearch, West Grove, PA) 64 and 10 μg 2.4G2 (BioXCell, West Lebanon, NH). Cells were labeled on wet ice in the dark. Samples 65 were run on a FACSCANTO II (Becton Dickinson, San Jose, CA) and data were analyzed using FlowJo 66 (Tree Star, Ashland, OR). 67 68 Detection and isotyping of serum paraproteins and measurement of serum cytokines Tompkins et al. – Supplemental Information 2 BCL2+IL6+ plasma cell tumors 69 Whole blood was collected from mice at necropsy, using heart puncture. Blood was transferred to 70 EDTA-coated Microtainer tubes (Becton Dickinson) and spun for 5 min at 14,000 RPM to obtain serum. 71 After centrifugation, serum was removed and frozen until the time of analysis. Serum protein 72 elecrophoresis was used to detect paraproteins (M-spikes). Serum proteins were fractionated on 73 Hydragel Protein(e) K20 gels using a Sebia elecrophoresis chamber (90 V constant; 40 min migration 74 time; 12±3 mA). Paraproteins were isotyped using the Mouse Immunoglobulin Isotyping ELISA from 75 BD Pharmingen according to the manufacturer’s recommendations. Modifications included dilution of 76 serum samples and HRP-labeled antibodies in blocking buffer containing 0.05% Tween, and using 77 HRP-labeled antibodies at 1:200. ELISA 96-well microplates were read at 450 nm using the Multiskan 78 Spectrum from Thermo Scientific. The Bio-Plex 200 system (BioRad, Hercules, CA) was used to 79 determine serum cytokine and chemokine levels, according to the manufacturer's instructions. Plates 80 were washed on a Bio-Plex Pro II device and analyzed on a Bio-Plex 200 reader. 81 82 In vivo bioluminescence imaging (BLI) analyses of tumor growth rates and tumor dissemination 83 patterns 84 Tumor formation was monitored using serial BLI on the IVIS100 system. Mice were anesthetized by 85 isoflurane inhalation maintained at 2.5% through a nose cone, and images were obtained in dorsal and 86 ventral presentations 5 min and 10 min after D-luciferin injection, respectively. Subsequently, a 87 rectangular region of interest was placed around the dorsal and ventral images for each mouse and 88 total photon flux (photons per second) was quantified using Living Image software v2.50 (Caliper Life 89 Sciences). Dorsal and ventral values were summed and compared to previous values to estimate the 90 whole-body tumor growth rate. Animals were observed twice weekly for adverse events associated 91 with tumor growth (e.g., >15% body weight loss, immobility, loss of grooming, paralysis), at which time 92 mice were euthanized. 93 94 Integrated FDG-PET and CT scanning and image analysis 95 We exactly followed the protocol described elsewhere 4. Briefly, for PET/CT scanning, mice were 96 anesthetized, administered 18F-FDG (8.65±2.7 MBq) via the lateral tail vein and placed prone in a 97 heated (36°C) multimodality chamber (M2M Imaging, Cleveland, OH) in the PET scanner’s gantry. 98 PET list mode data were acquired for 15 min, using an Inveon small-animal PET/CT/SPECT imaging 99 system (Preclinical Solutions, Siemens Healthcare Molecular Imaging, Knoxville, TN). In the same 100 workflow, a CT image was acquired for attenuation correction purposes. Images were reconstructed 101 using a 3D OP-MAP algorithm. Images were analyzed using PMOD v3.2 software (PMOD 102 Technologies, Zurich, Switzerland). Tompkins et al. – Supplemental Information 3 BCL2+IL6+ plasma cell tumors 103 Supplemental Figure Legends 104 105 Supplemental Figure 1: Bioluminescence imaging (BLI) of plasma cell neoplasms (PCNs) 106 genetically tagged with firefly luciferase (fLuc). 107 (A) BLI images of 2 tumor-bearing host mice (#19 and 20) in dorsal (left) and ventral (right) position 108 evaluated on days 96 (top) and 129 (bottom) following reconstitution with luciferase-transduced (Luc+) 109 B cells. Two additional tumor-bearing hosts (#8 and 12) reconstituted with donor-type B cells not 110 transduced with (Luc-) were included as control. The increased photon flux scales next to the heat 111 maps at the bottom reflect the incrased tumor burden at the later time point. 112 (B) Second set of host mice bearing either Luc+ tumors (#13, 17, 45 and 47) or a Luc- tumor (#14). 113 Images were taken on days 112 (top) and 202 (bottom) after B-cell reconstitution. 114 (C) Third set of host mice bearing either Luc+ tumors (#30 and 49) or Luc- tumors (#48). Images were 115 taken in a relatively short interval, on days 132 (top) and 144 (bottom) after B-cell reconstitution. The 116 reason for the lack of signal in case of mouse 30 remained unclear. 117 118 Supplemental Figure 2: Fast-onset tumors do not produce paraprotein. 119 (A) Presented is a serum protein electropherogram that contains samples from 6 different tumor- 120 bearing mice (lanes 2-7). M-spikes (paraproteins) are not detected. Included as a control is a serum 121 sample from a host mouse carrying a late-onset tumor (lane 1). The massive M-spike in that case is 122 indicated by red arrowhead pointing right. 123 (B) H&E-stained tissue section (100x) of a representative tumor included in lanes 2-7 of panel A. The 124 tumor was classified as diffuse large B-cell lymphoma (DLBCL) based on the following 125 histopathological criteria: large lymphoid tumor cells with nuclear size equal to or exceeding normal 126 macrophage nuclei and/or twice the size of a normal lymphocyte; diffuse growth pattern effacing normal 127 tissue architecture; and abundance of immunoblast- and centroblast-like cells. Note that hind limb 128 paralysis also occurred in some DLBCL-carrying mice, indicating that this diagnostic feature is not 129 specific for PCN in our adoptive-transfer mouse model of myeloma. 130 131 Supplemental Figure 3: Coexistent PCN and DLBCL in the bone marrow of the same mouse. 132 Shown is a H&E-stained bone marrow section (original magnification 100x) in which the approximate 133 borderline between the two tumors is indicated by a wavy yellow vertical line that bisects the image. 134 Blowups of the areas marked by yellow rectangles reveal distinct cytological features: immunoblast-like 135 cells in case of DLBCL (left) and atypical plasma cells with eccentric nuclei and large amounts of 136 cytoplasm in case of PCN (right). Tompkins et al. – Supplemental Information 4 BCL2+IL6+ plasma cell tumors 137 138 Supplemental Figure 4: Isotyping of paraproteins using ELISA. Serum M-spikes in 6 different host 139 mice carrying adoptive-transfer BCL2+IL6+ tumors were detected using protein electrophoresis (left; M- 140 spikes are indicated by red arrowheads) and then isotyped using ELISA (right). For the latter, serum 141 samples were logarithmically diluted, from 10-4 to 10-8, and probed with antibodies to immunoglobulin 142 heavy-chains and light-chains indicated vertically to the left of the 3 photographic images of 96-well 143 microplates. The 2 rightmost columns in these plates were used for positive and negative controls (Co) 144 supplied by the manufacturer. M-spikes in sera that contained single paraproteins were readily 145 isotyped: 2b in case of 1395, 1397 and 1403, and2a in case of 1162. Mouse 1161 harbored a 146 major and a minor M-spike, which were isotyped as and3. Because of its unusual electrophoretic 147 mobility near the -globulin fraction and our past experience with paraprotein typing in mice, it is likely 148 that the major M-spike indicates the IgM+ tumor. However, this has not been demonstrated. Because 149 usage of light-chains is unusual in mice, it is also possible that the IgM+ and IgG3+ tumors that 150 coexisted in mouse 1161 are clonally related, by virtue of isotype switching. But this has not yet been 151 shown. Mouse 1270 also harbored two M-spikes: one was isotyped as 2b (likely indicating the larger 152 spike) but it remained unclear whether the second spike was 1+ or +. 153 154 Supplemental Figure 5: Myeloma-like kidney disease. The kidneys in the host mice bearing 155 adoptive-transfer BCL2+IL6+ transgenic tumors exhibit morphologic changes consistent with myeloma 156 cast nephropathy. A representative example is shown. Numerous voluminous laminated eosinophilic 157 tubular casts are seen (top panel). The tubular cells appear flattened and show varying degrees of 158 necrosis and denudation of tubular basement membranes. There are significant acute (center panel, 159 left) and chronic (center panel, right) interstitial inflammatory cell infiltrates. The latter include 160 occasional multinucleated giant cells (bottom panel, right, and inset below) and focal collections of 161 neoplastic plasma cells (bottom panel, left, and inset below). 162 163 Supplemental Figure 6: Massive hypergammaglobulinemia and emerging M-spikes in double- 164 transgenic BCL2+IL6+ mice ~3-5 months of age. Serum protein electropherograms demonstrating 165 striking polyclonal elevations of immunoglobulins (hypergammaglobulinemia), emerging M-spikes in 2 166 cases (indicated by arrowheads pointing left in the top and bottom panels) and a pronounced M-spike 167 (monoclonal Ig a.k.a. paraprotein, indicated by arrowhead pointing up in the center panel) in one case 168 in a cohort of untreated BCL2+IL6+ mice (n = 15) ranging from ~3 months to ~5 months in age. A 169 serum sample from a 4-month-old inbred C mouse (not transgenic, not treated) was used as control. 170 The albumin and globulin fractions of serum proteins are labeled. Tompkins et al. – Supplemental Information 5 BCL2+IL6+ plasma cell tumors 171 172 Supplemental Figure 7: Flow-cytometric evaluation of a PCN that arose spontaneously in a 173 double-transgenic BCL2+IL6+ mouse ~5 months of age. Cells were gated on forward and side 174 scatter (not shown) and evaluated for expression of the B cell marker, B220, and the plasma cell (PC) 175 marker, CD138. B cells (B220+CD138+) were distinguished from PCs (B220+CD138+). Plasmablasts 176 (B220+CD138+) were not detected. Note the high frequency of PCs in the bone marrow (49%) relative 177 to the spleen (6.8%). However, in this case, another secondary (extramedullary) lymphoid tissue, the 178 perithymic lymph node, was as heavily infiltrated with neoplastic PCs as the bone marrow. 179 180 Supplemental Figure 8: Elevated serum levels of cyto- and chemokines. Presented are results of 181 Bio-Plex analyses of 3 groups of PCN-bearing BALB/c mice: (1) BCL2+IL6+ B-cell reconstituted mice 182 harboring myeloma-like tumors, as described in the main text (n = 6; center columns); (2) Myc+IL6+ B- 183 cell reconstituted mice containing peritoneal plasmacytomas (n = 6; left columns) that developed in 184 pristane-induced inflammatory granulomas, as described in a previous Letter 6 and (3) double- 185 transgenic BCL2+IL6+ mice harboring spontaneously arising tumors without adoptive B cell transfer or 186 any other manipulation (n = 7; right columns). Mean values and standard errors of the mean are 187 plotted. Statistical significance was determined with the help of the unpaired t test (in case of equal 188 variance) or the Mann-Whitney test (different variance). All 5 cytokines included in the figure have 189 been previously implicated in myeloma by other investigators. Thus, interleukin (IL)-5 and IL-13 are 190 TH2 cytokines that may promote myeloma cells in the bone marrow niche 7. Elevated IL-17, produced 191 by pro-inflammatory TH17 cells, promotes myeloma cell growth via a complex mechanism that includes 192 inhibition of immune functions 8. Monocyte chemotactic protein (MCP)-1 and tumor necrosis factor 193 alpha (TNF) play important roles in myeloma bone disease 9. See Supplemental Tables 1 and 2 for 194 additional cyto- and chemokines. More work is warranted before it can be decided which cyto- and 195 chemokines, if any, are important for the development of myeloma-like tumors in the BCL2+IL6+ 196 adoptive transfer model of human myeloma. 197 198 Supplemental Figure 9: General bone loss in lumbar vertebrae of host mice harboring BCL2+IL6+ 199 driven PCNs. CT images depicting bone thickness in false color are shown. The strong bone (white 200 to yellow) in the two normal mice (used as controls; left) compared to the weak bone (blue-to-red) in the 201 two tumor-bearing mice (right) can be readily appreciated upon visual inspection of images. Statistical 202 comparison of quantitative data (not shown) demonstrated a significant bone loss in diseased mice (p < 203 0.01). 204 Tompkins et al. – Supplemental Information 6 BCL2+IL6+ plasma cell tumors 205 206 Supplemental References 207 208 1. Strasser, A., Harris, A.W. & Cory, S. E mu-bcl-2 transgene facilitates spontaneous 209 transformation of early pre-B and immunoglobulin-secreting cells but not T cells. Oncogene 8, 1- 210 9 (1993). 211 2. 212 213 Proc.Natl.Acad.Sci.U.S.A. 86, 7547-7551 (1989). 3. 214 215 Suematsu, S. et al. IgG1 plasmacytosis in interleukin 6 transgenic mice. Morse, H.C., 3rd et al. Bethesda proposals for classification of lymphoid neoplasms in mice. Blood 100, 246-58 (2002). 4. Duncan, K. et al. (18)F-FDG-PET/CT imaging in an IL-6- and MYC-driven mouse model of 216 human multiple myeloma affords objective evaluation of plasma cell tumor progression and 217 therapeutic response to the proteasome inhibitor ixazomib. Blood Cancer J 3, e165 (2013). 218 5. 219 220 (2010). 6. 221 222 7. Wong, D. et al. Eosinophils and megakaryocytes support the early growth of murine MOPC315 myeloma cells in their bone marrow niches. PLoS One 9, e109018 (2014). 8. 225 226 Rosean, T.R. et al. The tumor microenvironment is the main source of IL-6 for plasma cell tumor development in mice. Leukemia (2014). 223 224 Doube, M. et al. BoneJ: Free and extensible bone image analysis in ImageJ. Bone 47, 1076-9 Prabhala, R.H. et al. Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma. Blood 115, 5385-92 (2010). 9. Liu, Z. et al. Bone marrow stromal cells derived MCP-1 reverses the inhibitory effects of multiple 227 myeloma cells on osteoclastogenesis by upregulating the RANK expression. PLoS One 8, 228 e82453 (2013). 229 Tompkins et al. – Supplemental Information 7